KR102179481B1 - Microorganism capable of reducing lipid content, and method and kit for identifying the same - Google Patents

Abstract

The present invention relates to a microorganism reducing the fat content of individuals, and to a method and kit for identifying the same, comprising a step of detecting at least one gene selected from the group consisting of LPL148-03160 gene of SEQ ID NO: 1, and LPL148-03166 gene of SEQ ID NO: 4.

Description

Microorganism capable of reducing lipid content, and method and kit for identifying the same

It relates to a microorganism Lactobacillus plantarum that reduces the fat content of a subject, a method and a kit for identifying the same.

Lactobacillus plantarum is a type of lactic acid bacteria as a bacillus contained in kimchi. Lactobacillus plantarum is also present in the intestine of animals.

Microorganisms in the genus Lactobacillus keep the intestinal pH acidic and inhibit the propagation of harmful bacteria such as E. coli and Clostridium. In addition, the microorganisms of the genus Lactobacillus are known to improve diarrhea and constipation, as well as synthesize vitamins and produce antibacterial substances.

On the other hand, it is known that subcutaneous injection of lipopolysaccharide endotoxin into mice induces obesity and insulin resistance through an inflammatory mediated pathway. In addition, it is known that the intestinal level of Enterobacter cloacae, which produces the lipopolysaccharide endotoxin, is associated with obesity (Na Fei et al., The ISME Journal (2013) 7, 880-884).

However, even according to the prior art, there is still a need for a microorganism of the genus Lactobacillus capable of reducing the fat content of an individual, and a method and kit for identifying it.

One aspect provides a method and kit for identifying a microorganism of the genus Lactobacillus capable of reducing fat content in adipocytes.

Another aspect provides a microorganism of the genus Lactobacillus capable of reducing fat content in adipocytes.

One aspect is the LPL148-03160 gene comprising the nucleotide sequence of SEQ ID NO: 1 and LPL148-03166 gene comprising the nucleotide sequence of SEQ ID NO: 4, which can reduce the fat content in an individual. Room microorganisms or extracts thereof are provided.

In the microorganism, the gene may be a foreign gene. That is, the microorganism may be a recombinant microorganism into which the gene has been introduced. The gene may be introduced into the genome of a microorganism. The gene may be introduced outside the genome of the microorganism. It is known to introduce foreign genes into Lactobacillus plantarum. The introduction may be, for example, by electroporation, transduction, or transformation. In addition, the microorganisms may be naturally separated microorganisms. That is, the microorganism may not be artificially manipulated. The microorganism may be, for example, Lactobacillus plantarum LMT1-48. The extract may be a supernatant remaining after dissolving microorganisms and removing precipitates by centrifugation.

The microorganism or its extract has antibacterial activity against E. cloacae that causes obesity, has acid resistance, has bile acid resistance, has activity to inhibit the differentiation of adipocytes into adipocytes , Inhibiting the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 among adipocytes, inhibiting the accumulation of fat among adipocytes, and when administered to an individual, body weight and fat in the body It may have one or more properties selected from the group consisting of reducing one or more selected from the group consisting of the volume of tissue, and reducing one or more selected from the group consisting of triglycerol and leptin content in blood when administered to an individual. have. The adipocyte progenitor cell may be 3T3-L1.

The acid resistance may be viable for 2 hours or more at pH 2.5 and 37° C. in MRS medium. Viable time is, for example, 4 hours or more, 10 hours or more, 15 hours or more, 24 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 2 hours to 24 hours, 2 hours to 48 hours, 2 hours To 60 hours, or 2 to 72 hours.

The bile acid resistance may be one capable of survival at 37° C. for 2 hours or more in an MRS medium containing 0.3% bile acid. Viable time is, for example, 4 hours or more, 10 hours or more, 15 hours or more, 24 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 2 hours to 24 hours, 2 hours to 48 hours, 2 hours To 60 hours, or 2 to 72 hours.

The microorganism or its extract may inhibit the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 among adipocytes. The inhibition is 10% or more, 15% or more, 20% or more, 25% or more of the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 compared to the case in which the microorganism or its extract is not present. % Or more, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more , 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or It may be to reduce 90% to 100%.

The microorganism or its extract may be one that inhibits the accumulation of fat in adipocytes. The inhibition is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more of fat accumulation based on the number or weight of the microorganisms compared to the case in which the microorganism or its extract is not present. % Or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% to 100%, 20% to 100% , 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% reduction.

When the microorganism or its extract is administered to an individual, it may reduce one or more selected from the group consisting of body weight and the volume of adipose tissue in the body. Compared to the case in which the microorganisms or extracts thereof are not present, fat accumulation is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45 % Or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% to 100%, 20% to 100% , 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% reduction.

When the microorganism or its extract is administered to an individual, it may reduce the amount of at least one selected from the group consisting of triglycerol and leptin in blood. The reduction is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more, based on the number or weight of microorganisms, compared to the case in which the microorganism or its extract is not present, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% to 100%, 20% to 100%, 30% To 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% reduction.

Another aspect provides a composition comprising the above-described microorganism or an extract thereof as an active ingredient.

The microorganisms or extracts thereof are as described above.

The composition has antimicrobial activity against E. cloacae, which causes obesity, has acid resistance, has bile acid resistance, has activity to inhibit the differentiation of adipocytes into adipocytes, adipocytes Inhibiting the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4, inhibiting the accumulation of fat in adipocytes, body weight and volume of adipose tissue in the body when administered to an individual It may have one or more properties selected from the group consisting of reducing one or more selected from the group consisting of, and reducing one or more selected from the group consisting of triglycerol in blood, and the amount of leptin when administered to an individual.

Therefore, the composition removes E. cloacae or inhibits proliferation, inhibiting the differentiation of adipocytes into adipocytes, among adipocytes, one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 Inhibits the expression of, inhibits the accumulation of fat in adipocytes, when administered to an individual, reduces one or more selected from the group consisting of body weight and the volume of adipose tissue in the body, and when administered to an individual, triglycerol in blood, And it can be used to reduce one or more selected from the group consisting of the amount of leptin. These uses may be used alone or in combination thereof.

The composition may be food or pharmaceutical. The composition may contain a food pharmaceutically or pharmaceutically acceptable carrier.

In the composition, the microorganism or extract may be included in a "food effective amount" or a "therapeutically effective amount". In the above composition, "therapeutically effective amount" means an amount sufficient to exhibit a therapeutic effect when administered to an individual in need of treatment. The term “treatment” means treating a disease or medical condition, eg, obesity disease, in an individual, eg, a mammal, including humans, including: (a) preventing the occurrence of the disease or medical condition, Ie, prophylactic treatment of the patient; (b) alleviation of the disease or medical condition, ie, causing elimination or recovery of the disease or medical condition in the patient; (c) inhibition of the disease or medical condition, ie slowing or stopping the progression of the disease or medical condition in the subject; Or (d) alleviating the disease or medical condition in the subject. The "effective amount" can be appropriately selected by a person skilled in the art. The "effective amount" may be 0.01mg to 10,000mg, 0.1mg to 1000mg, 1mg to 100mg, 0.01mg to 1000mg, 0.01mg to 100mg, 0.01mg to 10mg, or 0.01mg to 1mg.

The composition can be administered orally. Accordingly, the composition may be formulated in various forms such as tablets, capsules, aqueous solutions or suspensions. In the case of tablets for oral use, excipients such as lactose and corn starch, and lubricants such as magnesium stearate may be usually added. In the case of capsules for oral administration, lactose and/or dried corn starch may be used as a diluent. If oral aqueous suspensions are required, the active ingredient can be combined with emulsifying and/or suspending agents. If necessary, certain sweetening and/or flavoring agents can be added.

Another aspect provides a method for reducing the fat content in adipocytes, comprising; contacting the microorganism or its extract with adipocytes.

In the above method, the contacting may be to cultivate a microorganism or an extract thereof in an adipocyte in a medium. The method may be an in vitro or in vivo method.

Another aspect provides a method for reducing the fat content of an individual, comprising; administering the microorganism or an extract thereof to the individual.

In the above method, a person skilled in the art can appropriately select an administration route according to the condition of the patient during administration. The administration may be oral or topical.

In the above method, the dosage varies according to various factors such as the patient's condition, the route of administration, and the doctor's judgment, as described above. Effective dosages can be estimated from dose-response curves obtained in vitro or in animal model tests. The proportion and concentration of the compound of the present invention present in the composition to be administered may be determined according to chemical properties, route of administration, therapeutic dosage, and the like. The dosage may be administered to an individual in an effective amount of about 1 μg/kg to about 1 g/kg per day, or about 0.1 mg/kg to about 500 mg/kg per day. The dosage may vary depending on the age, weight, sensitivity, or symptoms of the individual. The subject may be a non-human mammal.

Another aspect is a detection reagent that specifically binds to at least one selected from the group consisting of the LPL148-03160 gene comprising the nucleotide sequence of SEQ ID NO: 1, the LPL148-03166 gene comprising the nucleotide sequence of SEQ ID NO: 4, and an expression product thereof. to provide.

The gene is a gene specifically present in Lactobacillus plantarum microorganisms capable of reducing fat content in individuals. The LPL148-03160 gene has a nucleotide sequence of SEQ ID NO: 1 and may encode a polypeptide having an amino acid sequence of SEQ ID NO: 9. The product of the gene may function as a histidine utilization repressor. As the LPL148_03160 gene-specific primer set, a primer set (LPL148_03160_1) of SEQ ID NOs: 2 and 3 was prepared.

The LPL148_03166 has a nucleotide sequence of SEQ ID NO: 4 and may encode a polypeptide having an amino acid sequence of SEQ ID NO: 10. The product of the gene may function as a mannose-6-phosphate isomerase. As LPL148_03166 gene-specific primer sets, primer sets of SEQ ID NOs: 5 and 6 (LPL148_03166_6) and primer sets of SEQ ID NOs: 7 and 8 (LPL148_03166_8) were prepared.

The detection reagent may be a polynucleotide that specifically binds to the nucleotide sequence of SEQ ID NO: 1 or a complementary nucleotide sequence thereof, or the nucleotide sequence of SEQ ID NO: 4 or a complementary nucleotide sequence thereof. The detection reagent may include one or more of the polynucleotides of SEQ ID NOs: 2, 3, 5, 6, 7, and 8.

The detection reagent includes a polynucleotide of SEQ ID NO: 2 and a polynucleotide of SEQ ID NO: 3; The polynucleotide of SEQ ID NO: 5 and the polynucleotide of SEQ ID NO: 6; And the polynucleotide of SEQ ID NO: 7 and the polynucleotide of SEQ ID NO: 8; may include one or more of the polynucleotide set consisting of.

The detection reagent may be an antibody that specifically binds to the expression product. The expression product may be at least one selected from the group consisting of the polypeptide of SEQ ID NO: 9 and the polypeptide of SEQ ID NO: 10.

The detection reagent may be detectable or labeled with a label capable of generating a detectable label. The label may be a fluorescent material, a radioactive material, or an enzyme. The label may be a fluorescent protein such as GFP, or an enzyme capable of changing a detectable signal for a substrate such as alkaline phosphatase.

Another aspect provides a composition for identifying Lactobacillus plantarum capable of reducing fat content among individuals comprising the detection reagent.

The composition may contain an excipient, a diluent or a carrier. The carrier may be a buffer, a solvent, or a stabilizer.

Another aspect provides a kit for identifying Lactobacillus plantarum capable of reducing fat content among individuals comprising the detection reagent.

The kit may include instructions explaining the process of using the detection reagent to identify Lactobacillus plantarum capable of reducing fat content in an individual.

In the composition or kit, the Lactobacillus plantarum may be one capable of reducing the fat content in adipocytes.

Another aspect is a method for identifying a Lactobacillus plantarum capable of reducing the fat content of a subject in a sample comprising; contacting the detection reagent with a sample containing Lactobacillus plantarum or a product derived therefrom. to provide.

The Lactobacillus plantarum may be one capable of reducing fat content in adipocytes. The product derived therefrom may be a product isolated or amplified from the Lactobacillus plantarum. The product derived therefrom may be a nucleic acid or a protein.

The method may further include contacting the detection reagent with a sample that does not contain or is determined to contain Lactobacillus plantarum or a product derived therefrom.

The method may further include measuring a signal from a reaction product of the detection reagent and Lactobacillus plantarum or a product derived therefrom.

The method includes an increase in the signal from the reaction product of the detection reagent and the Lactobacillus plantarum or a product derived therefrom compared to a sample not containing the Lactobacillus plantarum or a product derived therefrom. It may further include a step of determining that the plantar room exists.

The step of contacting may include one or more of performing a polymerase chain reaction (PCR), performing a nucleic acid hybridization reaction, and performing an antigen-antibody reaction.

The method includes, for example, the polynucleotide of SEQ ID NO: 2, the polynucleotide of SEQ ID NO: 3, the polynucleotide of SEQ ID NO: 5, the polynucleotide of SEQ ID NO: 6, the polynucleotide of SEQ ID NO: 7 and the polynucleotide of SEQ ID NO: 8; Amplifying the nucleic acid by contacting one or more of the polynucleotides with a nucleic acid derived from Lactobacillus plantarum in a sample; And it may be to include the step of measuring the level of the amplification product. The nucleic acid may be a genome of Lactobacillus plantarum, mRNA, which is an expression product of the gene, or cDNA prepared therefrom.

The term “amplification” refers to increasing the copy number of a target sequence or its complementary sequence. Amplification can be accomplished by any method known in the art. Amplification of nucleic acids includes methods that require multiple cycles during amplification or methods performed at a single temperature. Examples of cycling techniques include those requiring thermal cycling. Methods requiring thermal cycling include polymerase chain reaction (PCR). PCR is known in the art. PCR usually involves denaturing double-stranded DNA into single-stranded DNA by heat denaturation, and annealing a primer to the single-stranded DNA; And synthesizing a complementary strand from the primer. The isothermal amplification method is a method in which a single temperature or the main aspect of the amplification process is performed at a single temperature. In contrast to PCR, which heats the reaction product to allow additional primers to bind to separate the double strands, the isothermal method relies on strand displacing polymerase to separate the double strands and recopy the template. do. The isothermal method can be divided into a method that relies on substitution of a primer to initiate repetitive template copying and a method that relies on continuous reuse or novel synthesis of a single primer molecule. Methods that rely on substitution of primers include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA), and loop-mediated amplification. It includes a method selected from the group consisting of (loop mediated amplification: LAMP). Methods that rely on continuous reuse or novel synthesis of single primer molecules include methods selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA).

Measuring the level of the amplification product may include electrophoresis of the amplification product and then binding it with a double-stranded nucleic acid-specific dye such as EtBr.

The method includes, for example, the polynucleotide of SEQ ID NO: 2 and the polynucleotide of SEQ ID NO: 3; The polynucleotide of SEQ ID NO: 5 and the polynucleotide of SEQ ID NO: 6; And amplifying the nucleic acid by contacting at least one of the polynucleotide set consisting of the polynucleotide of SEQ ID NO: 7 and the polynucleotide of SEQ ID NO: 8 with a nucleic acid derived from Lactobacillus plantarum in a sample; And it may be to include the step of measuring the level of the amplification product. The nucleic acid may be a genome of Lactobacillus plantarum, mRNA, which is an expression product of the gene, or cDNA prepared therefrom. The amplification may be PCR.

The microorganism according to one aspect or an extract thereof may be used to reduce the fat content in adipocytes or to reduce the fat content in individuals.

A composition for use in reducing the fat content in adipocytes according to another aspect, or a composition for use in reducing the fat content in an individual, a composition for use in reducing the fat content in adipocytes, or reducing the fat content in an individual Can be used to make.

According to the method of reducing the fat content of adipocytes or the method of reducing the fat content of an individual according to another aspect, the fat content of the adipocytes can be efficiently reduced or the fat content of the individual can be effectively reduced.

According to the detection reagent according to another aspect, a composition and a kit including the same, it can be used to efficiently identify Lactobacillus plantarum microorganisms capable of reducing fat content in adipocytes.

According to the method for identifying Lactobacillus plantarum that can reduce the fat content of individuals in the sample according to another aspect, it is possible to efficiently identify Lactobacillus plantarum that can reduce the fat content of individuals in the sample.

1 shows a scanning electron microscope (Scanning Electron Microscopy, SEM) image of the selected lactic acid bacteria.
2 is a view showing the number of cells of E. cloacae KCTC 1685 cultured in a medium containing a culture medium corrected to the same pH as the lactic acid bacteria culture medium.
3 is a diagram showing the number of selected microorganisms attached to intestinal epithelial cells.
4 is a diagram showing electron micrographs (FIGS. 4A to 4C) of adipocytes of 3T3-L1 adipocytes cultured in the presence of a culture medium of the selected microorganism.
5 shows the results of confirming the expression of PPARν, C/EBPα, FAS, and FABP4 genes related to obesity in 3T3-L1 adipocytes cultured in the presence of a culture medium of the selected microorganism at the mRNA level.
6 is a diagram showing the change in body weight over time when the selected microorganisms are administered in obesity-induced mice.
7 is a diagram showing the volume of body fat when a selected microorganism is administered in an obesity-induced mouse.
Figure 8 is a case of administering selected microorganisms in obesity-induced mice, triglycerides in serum (Figure 8a), total cholesterol (Figure 8b), high-density lipoprotein (HDL) (Figure 8c), low-density lipoprotein (LDL) (Figure 8d) , Leptin (FIG. 8E), and adiponectin (FIG. 8F) is a diagram showing the results of measuring the content.
9 is a diagram showing the result of classifying the intestinal microorganisms in the feces at the class level when the selected microorganisms are administered in obesity-induced mice.
FIG. 10 shows the three species-gene-specific primer set positions selected in this example, PCR was performed using the same, and the result of electrophoresis analysis of the product.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Clocae known to cause obesity in the following examples Microorganisms of the genus Lactobacillus that inhibit growth were selected. The indicator bacteria used in the experiment were Enterobacter cloacae (E. cloacae KCTC 1685) (hereinafter also referred to as'indicator bacterium 1'), Enterobacter cloacee subspecies Cloace ( Enterobacter). subsp. cloacae KCTC 2361) (hereinafter also referred to as'indicator 2'), which was sold and used by the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center. In addition, lactic acid bacteria was identified and isolated directly from traditional foods, such as Kimchi and salted fish, were screened through a bacterial growth inhibitory effects of obesity screening using these lactic acid bacteria, i.e. strains having a high antibacterial activity against the bacteria of which obesity, Lactobacillus plantarum LMT1- 48 (hereinafter also referred to as'LMT1-48') could be secured.

This strain was deposited with the Korea Cell Line Bank (KCTC), microbial metabolism characterization (API kit), morphological analysis using an electron microscope, stability test in the intestine through acid resistance and bile resistance analysis, and adipocyte differentiation of adipocytes. The effect of reducing body fat was verified through ability verification and animal testing. It will be described in detail below.

Example 1. Isolation and identification of strains

1. Isolation and selection of strains

Kimchi, which was used to isolate lactic acid bacteria, was stored at 4°C. For the isolation of total lactic acid bacteria from kimchi, it was plated on MRS medium (Difco Co., USA) and cultured at 30°C. The pretreatment method of kimchi is to take 20g of kimchi at 4℃ aseptically, dilute it with 180 ml of 0.85% NaCl solution, homogenize for 5 minutes with a stomacher, and then sterilize a tube containing 9 ml of 0.85% NaCl solution. Samples were prepared by stepwise dilution in tubes. After that, it was plated on MRS plate and cultured at 37°C for 2-3 days, and the colonies that appeared were separated by morphology and color and purified again. The isolated colonies were cultured in MRS liquid medium of pH 6.8 at 37° C. for 24 hours to select 128 colonies whose pH decreased to 4.5 or less. Subsequently, it was identified by 16S rRNA sequencing, and lactic acid bacteria that inhibit the growth of obesity-related bacteria were selected out of the isolated 128 lactic acid bacteria. SEQ ID NO: 11 is the nucleotide sequence of the 16S rRNA gene of L. plantarum LMT1-48.

In order to select the lactic acid bacteria that inhibit the growth of obesity-related bacteria among the 128 lactic acid bacteria isolated above, indicator bacteria 1, which are obesity-related bacteria, and Enterobacter cloase subspecies Cloase were added to the MRS agar plate at 1x10 ⁶ CFU/ml. 5 μl of lactic acid bacteria were instilled and cultured at 37° C. for 24 hours to select 42 lactic acid bacteria having inhibitory ability as shown in Table 1.

Thereafter, the first 42 lactic acid strains selected were placed on a disc paper in the same manner and an antimicrobial activity comparison experiment was conducted. At this time, in order to select each of the lactic acid bacteria strains with the best antibacterial activity at each species level, only the culture medium (OD ₆₀₀ ≒2.0) was collected, 100 µl was added dropwise, and cultured at 37°C for 24 hours, followed by secondary selection. Finally, one type of lactic acid bacteria having the best ability to inhibit the growth of obese bacteria was selected.

number Lactobacillus (Strains) Indicator strain growth inhibition ring (mm) E. cloacae
KCTC 1685 E. subsp. Cloacae KCTC 2361 One Lactobacillus plantarum LMT1-2 15 15 2 Lactobacillus plantarum LMT1-3 13 13 3 Lactobacillus plantarum LMT1-13 12 12 4 Lactobacillus plantarum LMT1-33 12 12 5 *Lactobacillus plantarum LMT1-48 18 18 6 Lactobacillus plantarum LMT3-1 14 14 7 Lactobacillus plantarum LMT3-26 17 18 8 Lactobacillus plantarum LMT3-66 12 12 9 *Lactobacillus paracasei LMT1-30 16 16 10 * Pediococcus acidilactici LMT2-46 18 18 11 Lactobacillus casei LMT1-31 11 11 12 Lactobacillus casei LMT1-71 14 13 13 Lactobacillus sakei LMT1-21 11 11 14 Lactobacillus sakei LMT1-36 13 14 15 Lactobacillus sakei LMT1-66 15 15 16 Lactobacillus brevis LMT1-46 12 12 17 Lactobacillus brevis LMT1-73 12 12 18 Leuconostoc lactis LMT2-35 12 12 19 Lactobacillus parabuchneri LMT1-47 11 11 20 Weissella cibaria LMT2-10 14 13 21 Weissella halotolerans LMT2-48 11 11 22 Pediococcus pentosaceus LMT1-1 14 13 23 Pediococcus pentosaceus LMT1-63 14 14 24 Pediococcus pentosaceus LMT2-64 14 14 25 Pediococcus pentosaceus LMT2-68 12 12 26 Pediococcus pentosaceus LMT3-5 12 12 27 Pediococcus pentosaceus LMT3-41 13 13 28 Leuconostoc mesenteroides LMT1-8 14 13 29 Leuconostoc mesenteroides LMT1-26 13 13 30 Leuconostoc mesenteroides LMT1-50 13 13 31 Leuconostoc mesenteroides LMT2-4 11 12 32 Leuconostoc mesenteroides LMT1-75 11 11 33 Leuconostoc mesenteroides LMT1-79 12 12 34 Lactobacillus fermentum LMT3-3 12 12 35 Lactobacillus fermentum LMT3-67 12 12 36 Leuconostoc citreum LMT2-9 14 14 37 Enterococcus faecalis LMT2-19 11 11 38 Enterococcus faecium LMT2-13 12 12 39 Enterococcus faecium LMT2-14 11 12 40 Enterococcus faecium LMT3-60 11 11 41 Enterococcus faecium LMT3-61 11 11 42 Lactococcus lactis subsp. Cremoris LMT2-37 12 12

As a result of examining the antibacterial activity of the selected lactic acid bacteria, it was possible to measure the antibacterial activity by the diameter of a clear zone that was clearly created around the disk. The culture medium of L. plantarum LMT1-48 was 18 mm, showing antimicrobial activity against indicator bacteria 1 and Enterobacter cloase subspecies Cloase, and it was confirmed that it had minimal antimicrobial activity. Therefore, the lactic acid bacteria culture medium of the present invention has excellent antibacterial activity against obesity-related bacteria.

2. Identification of the selected strain

(One) Mycological Characteristic analysis

The selected lactic acid bacteria were cultured in MRS plate medium (Difco, USA), and the morphology of colonies was observed. The colony forms in the MRS plate medium of the selected lactic acid bacteria are shown in Table 2 below.

1 shows a scanning electron micrograph of the selected lactic acid bacteria. As shown in Figure 1, rod-shaped bacillus and spherical shape similar to typical Lactobacillus genus.

LMT1-48 shape circle size 1mm color cream color Opacity opacity bump Convex surface lubricity Aerobic growth + Anaerobic growth +

(2) 16S rDNA analysis

For the PCR reaction of the isolated lactic acid bacteria, the universal bacterial primer was commissioned to Macrogen Co., Ltd. using 27F (SEQ ID NO: 12) and 1492R (SEQ ID NO: 13) to amplify the 16S rRNA gene, which is the base conserving sequence of bacteria. Analysis was carried out. The results were analyzed using NCBI blast (http://www.ncbi.nlm.nih.gov/).

The present inventors named LMT1-48 lactic acid bacteria " Lactobacillus plantarum ( Lactobacillus plantarum ) LMT1-48 (Accession No.: KCTC13024BP)" and this in the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology. Deposited on May 13, 2016.

(3) selected Lactic acid bacteria Sugar fermentation properties

Sugar fermentation characteristics were investigated according to the experimental method of the supplier using the API 50 CHL kit (Biomerieux, France). The sugar fermentation properties of the lactic acid bacteria L. plantarum LMT1-48 are shown in Table 3 below.

Party LMT1-48 Glycerol - Erythritol - D-Arabinose - L-Arabinose - D-Ribose + D-Xylose - L-Xylose - D-Adonitol - Methyl-β-D-Xylopyranoside - D-Galactose + D-Glucose + D-Fructose + D-Mannose + L-Sorbose - L-Rhamnose - Dulcitol - Inositol - Mannitol + D-Sorbitol + Methyl-α-D-mannopyranoside + Methyl-α-D-glucopyranoside - N-Acetylglucosamine + Amygdalin + Arbutin + Esculin + Salicin + D-Cellobiose + D-Maltose + D-Lactose + D-Melibiose + D-Saccharose + D-Trehalose + Inulin - D-Melezitose + D-Raffinose + Amidon - Glycogen - Xylitol - Gentiobiose + D-Turanose + D-Lyxose - D-Tagatose - D-Fucose - L-Fucose - D-Arabitol - L-Arabitol - Potassium Gluconate + Potassium 2-Ketogluconate - Potassium 5-Ketogluconate -

Table 3 is a table showing the analysis results of sugar fermentation pattern of lactic acid bacteria according to the present invention.

3. Functionality

(1) Measurement of minimum antibacterial activity of lactic acid bacteria culture medium

In order to measure the minimum antimicrobial activity of the selected lactic acid bacteria culture medium having excellent antibacterial activity, it was measured using an agar well diffusion assay. That is, the culture supernatant (OD ₆₀₀ ≒2.0) was dropped on the MRS agar plate to which the indicator strain was added at 1×10 ⁶ CFU/ml, and cultured at 37° C. for 24 hours, and then the formation of an inhibitory ring was observed. The activity titer was expressed as an activity unit (AU) per ml of the culture medium. That is, the reciprocal of the maximum dilution factor for forming an inhibitory ring was taken by sequentially diluting the culture solution twice, and this value was multiplied by the conversion factor converted to 1 ml, and expressed as AU/ml as shown in Table 4 below.

Indicated strain Antibacterial activity of LMT1-48 (AU/ml) E. cloacae KCTC 1685 1600 E. subsp. cloacae KCTC 2361 1600

As a result of measuring the minimum antibacterial activity of the lactic acid bacteria culture medium of the present invention, the culture medium of L. plantarum LMT1-48 showed the minimum antibacterial activity at 1600AU/mL.

(2) Confirmation of dependent antimicrobial activity by lactic acid bacteria culture medium components

The final pH of the selected lactic acid bacteria culture medium having excellent antibacterial activity (OD ₆₀₀ ≒2.0) was measured and shown in Table 5, and the MRS liquid medium was adjusted to be the same as the pH of the lactic acid bacteria culture medium with HCl. The lactic acid bacteria culture medium and the pH-adjusted MRS liquid medium were mixed so that the ratio of the lactic acid bacteria culture medium was 0%, 5%, 10%, 15%, and 20%, and the indicator bacteria cultured in MRS liquid medium at 37°C for 24 hours. 1 was inoculated so that the initial number of bacteria was 1×10 ⁶ CFU/ml, and cultured at 37°C for 24 hours. Samples after cultivation were recovered from lactic acid bacteria culture solutions of different %, diluted in MRS liquid medium, plated on MRS plate medium, incubated at 37°C for 24 hours, and then the number of colonies on the plate medium was counted and shown in FIGS. 2 and 5 Done. Figure 2 is a diagram showing the number of cells of KCTC 3108 ^T cultured in a medium containing a lactic acid bacteria culture medium of varying amounts.

E. cloacae (CFU) Addition rate (%) HCl calibration medium (pH 3.96) Lactobacillus culture filtrate (pH 3.96) 100:0(0) 2.4x10 ¹⁰ 2.4x10 ¹⁰ 95:5(5) 2.0x10 ¹⁰ 1.4x10 ⁹ 90:10(10) 9.9x10 ⁹ 3.0x10 ⁷ 85:15(15) 1.5x10 ⁸ 1.4x10 ⁸ 80:20(20) 3.6x10 ⁵ 5.5x10 ⁵

As a result, in the liquid medium containing 5% of the lactic acid bacteria culture medium of L. plantarum LMT1-48, the indicator bacteria 1 were inhibited by 93%, respectively, and the MRS liquid medium pH-corrected with HCl solution was the same as the pH of the final culture medium of lactic acid bacteria. In the medium added with 5%, the growth inhibition of indicator bacteria 1 was relatively decreased.

Therefore, it was confirmed that the growth of indicator bacteria 1 was inhibited by pH, and particularly important, it was confirmed that the growth of indicator bacteria 1 was specifically inhibited by organic acids contained in the lactic acid bacteria culture medium, not by simple acidity (pH). (Fig. 2 and Table 5).

4. Stability

(1) of lactic acid bacteria Acid resistance Research

In order for lactic acid bacteria to exert its efficacy as a probiotic in the intestine, it must pass through a low pH stomach after ingestion. In order to investigate the acid resistance of lactic acid bacteria, inoculate the sterilized MRS liquid medium and incubate for 16 hours at 37°C, and then adjust the pH to 2.5 with HCl and inoculate 1% of the lactic acid bacteria into the sterilized MRS liquid medium for 2 hours at 37°C. During incubation. Samples immediately after inoculation of lactic acid bacteria and after 2 hours of culture were collected, diluted in MRS liquid medium, plated on MRS plate medium, and cultured at 37°C for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of lactic acid bacteria. The number of lactic acid bacteria in the experimental group, which was performed in the same manner in the neutral MRS liquid medium in which the pH is not adjusted, and the number of lactic acid bacteria after 2 hours incubation in the pH 2.5 MRS liquid medium, were compared and shown in Table 6 below.

Lactobacillus (CFU/ml) LMT1-48 Control 5.1x10 ⁹ pH 2.5 MRS 3.5x10 ⁸

As a result, the lactic acid bacteria selected in the present invention were very resistant to acidity at pH 2.5. The characteristic of these lactic acid bacteria is that the proper number of lactic acid bacteria is maintained at a pH lower than pH 3, which is close to the physiological pH of the stomach.Therefore, the number of surviving lactic acid bacteria is high even at a low pH due to gastric acid secretion.

(2) of lactic acid bacteria Bile resistance Research

In order to investigate the internal bile of lactic acid bacteria, an experiment was conducted by the following method. Lactobacillus was inoculated in sterilized MRS liquid medium and incubated for 24 hours at 37°C. Considering that the concentration of bile salts in the small intestine is around 0.1%, the lactic acid bacteria 1 were added to MRS liquid medium containing 0.3% bile salts. % Was inoculated and incubated at 37° C. for 2 hours. Samples immediately after inoculation of lactic acid bacteria and after 2 hours of culture were collected, diluted in MRS liquid medium, plated on MRS plate medium, and cultured at 37°C for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of lactic acid bacteria. Table 7 shows the number of lactic acid bacteria in the experimental group performed in the same manner in the MRS liquid medium without 0.3% bile salt and the number of lactic acid bacteria after 2 hours incubation in the MRS liquid medium containing 0.3% bile salt.

Lactobacillus (CFU/ml) LMT1-48 Control 5.1x10 ⁹ 0.3% bile salt 4.8x10 ⁸

As a result, since the lactic acid bacteria selected in the present invention maintained an appropriate number of lactic acid bacteria even at 0.3% higher than 0.1% similar to the actual concentration in the intestine, the lactic acid bacteria can be a basis for confirming that they can sufficiently survive in the intestine of humans or animals. I can.

(3) Investigation of intestinal fixation of lactic acid bacteria

The Caco-2 cell line, a human epithelial colorectal adenocarcinoma cell, purchased from the Korea Cell Line Bank was used to confirm the degree of fixation in the intestinal cells. Caco-2 cells were Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and were cultured at 5% CO ₂ and 37°C. L. plantarum LMT1-48 cultured in MRS medium was washed with Phosphate Buffered Saline (PBS) and then suspended in DMEM medium to which antibiotics were not added, and this was suspended in a 96-well cell culture plate (Corning, USA). ) To Caco-2 cells formed with a single layer in 1×10 ⁷ CFU, and cultured for 2 hours at 5% CO ₂ and 37°C. To remove lactic acid bacteria that could not adhere to Caco-2 cells, wash them 5 times with PBS, remove the lactic acid bacteria attached with 100 µl of 0.1% Triton X-100, and spread them on MRS solid medium for 24 hours at 37°C. After culturing during, the colony number on the plate medium was counted to investigate the intestinal fixation of lactic acid bacteria.

The results are shown in FIG. 3. FIG. 3 is a diagram showing the number of selected three microorganisms attached to intestinal epithelial cells. As shown in Figure 3, it was confirmed that the lactic acid bacteria L. plantarum LMT1-48 of the present invention has a fixation power in Caco-2, an intestinal epithelial cell of about 106.3%.

Example 2. Effect of strain administration on obesity

1. Securing cell extract of lactic acid bacteria

L. Plantarum LMT1-48 was cultured in MRS medium at 37° C. for 24 hours. The cells were centrifuged and rinsed with PBS to remove remaining media components. After resuspending the lactic acid bacteria from which the medium was removed in a volume ratio of lactic acid bacteria: PBS: beads = 1: 2: 1, a protein degradation inhibitor 100 mM phenylmethylsulfonyl fluoride (PMSF) was added, and a bead beater was used. The cells were lysed using. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was filtered through a 0.2 μm filter to quantify protein, and 200 μg/ml of L. plantarum LMT1-48 cell extract was used in the experiment.

2. Measurement of 3T3-L1 adipocyte differentiation inhibitory ability by selected lactic acid bacteria

(1) 3T3-L1 adipocyte differentiation induction

DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) was used for 3T3-L1 cells, mouse embryonic fibroblasts purchased from the Korea Cell Line Bank (KCLB). % CO ₂ , cultured at 37°C.

The procedure for differentiating 3T3-L1 cells into adipocytes is as follows. 2 days after 3T3-L1 cells grew confluently (day 0), the culture medium was added to 10% FBS, 5 µg/ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma , USA) and 1 μM dexamethanesone (Sigma, USA) was exchanged with a DMEM medium, that is, a differentiation medium. After 2 days (day 2), the culture was performed after exchange with DMEM medium containing 10% FBS and 5 μg/ml insulin. After 2 days (day 4), the medium was exchanged with only 10% FBS and cultured. After 4 days (day 8), more than 90% of the 3T3-L1 cells were completely differentiated into adipocytes. Until 3T3-L1 cells completely differentiate into adipocytes (day 0 to day 8), they are termed maturing 3T3-L1 pre-adipocytes, and after fully differentiated into adipocytes ( After day 8), they are called mature 3T3-L1 adipocytes.

(2) 3T3-L1 In fat cells Fat accumulation inhibition ability measurement (Oil-red-O staining assay)

Each lactic acid bacterium cell extract was added together at 200 μg/ml at the time point (day 0) when the differentiation medium was added to the cultured 3T3-L1 adipocytes. After 6 days (day 6), the resulting maturing 3T3-L1 pre-adipocyte culture solution was washed 3 times with PBS to remove the culture solution, and 10% formalin (Merck millipore, Germany) The cells were fixed by adding for 1 hour. Thereafter, the oil red O (Sigma, USA) solution was reacted with the cells for 1 hour, washed with distilled water, and stained with fat bulbs, and the results were observed under a microscope (FIGS. 4A to 4C ).

The results are shown in FIG. 4. 4 is a diagram showing electron micrographs (FIGS. 4A to 4C) of adipocytes of 3T3-L1 adipocytes cultured in the presence of a culture medium of the selected microorganism. As shown in Figure 4, compared to the control group treated with the L. plantarum LMT1-48 cell extract reduced the degree of fat accumulation of 49.7%.

(3) 3T3-L1 intracellular obesity related Biomarker Gene expression Significant Measure the difference

Representative genes involved in the process of differentiating 3T3-L1 cells into adipocytes include PPARν, C/EBPα, FAS, and FABP4. Real-time PCR was performed using the cell extract of L. plantarum LMT1-48 to confirm whether the 3T3-L1 adipocyte differentiation inhibitory effect was indicated by suppressing the mRNA expression of the gene.

At the time point when the cultured 3T3-L1 adipocytes were treated with the differentiation medium (day 0), the lactic acid bacteria cell extract was treated with 200 µg/ml. After 6 days (day 6), the resulting mature 3T3-L1 adipocytes were washed 3 times with PBS to remove the lactic acid bacteria cell extract, and RNA was extracted using Trizol (Thermo scientific, USA). Thereafter, after obtaining DNA complementary to RNA using RocketScript Cycle RT Premix (Bioneer, South Korea), expression of adipocyte differentiation-related genes was confirmed using SYBR green (Takara, Japan). The results are shown in FIG. 5. 5 shows the results of confirming the expression of PPARν, C/EBPα, FAS, and FABP4 genes related to obesity in 3T3-L1 adipocytes cultured in the presence of a culture medium of the selected microorganism at the mRNA level.

As shown in Figure 5, compared to the control group treated with lactic acid bacteria extract, the mRNA expression levels of the PPARν, C/EBPα, FAS, and FABP4 genes were 78.6% and 78.8% respectively in the group treated with L. plantarum LMT1-48 cells. , 68.8%, and 85.4% were observed to be inhibited.

In addition, in the lactic acid bacteria culture solution treatment group, the mRNA expression levels of the PPARν, C/EBPα, FAS, and FABP4 genes were 21.9%, 50.1%, 38.2%, and 47% respectively in the L. plantarum LMT1-48 culture solution treatment group compared to the control group. It was confirmed that the decrease was inhibited by an amount.

Table 8 shows the primers used in real-time PCR, and the mRNA expression of the GAPDH gene is an internal control.

number gene primer Sequence number One
PPARν
Forward 14 Reverse 15 2
C/EBPα
Forward 16 Reverse 17 3
FAS
Forward 18 Reverse 19 4
FABP4
Forward 20 Reverse 21 5
GAPDH
Forward 22 Reverse 23

3. Obese bacteria E. on cloacae Caused by obesity C57BL In vivo efficacy evaluation of selected lactic acid bacteria in /6J mouse model

(One) C57BL Obesity induction and lactobacillus treatment in /6J mice

The animals used in the experiment were 5-week-old C57BL/6J mice (male, 20-22g) inducing obesity with a high fat diet, purchased from Jackson Laboratory (Bar Harbor, USA) and fed for 1 week (Teklad global diet 2014, Envigo , USA) to adapt to the environment. Afterwards, lactobacilli were administered directly into the stomach for 2 weeks using a sonde for oral administration once a day for the group to which lactic acid bacteria were administered, and after feeding the lactic acid bacteria, a high fat diet (Teklad custom diet TD.93075, Envigo, USA) and obese bacteria were administered. Induction of obesity was induced by administration of E. cloase in the same manner for 8 weeks.

L. plantarum LMT1-48 was orally administered at 1.0 x 10 ⁹ CFU / day and E. chlorase 1.0 x 10 ⁸ CFU / day. The group (n=8) consisted of a total of 7 groups as shown in Table 9 below.

P1 Normal dietary group P2 High fat diet group P3 High fat diet + Enterobacter cloase administration group P4 High fat diet + Enterobacter cloase + L. plantarum LMT1-48 administration group

Body weight and diet were measured once a week, and after the end of the experiment period, animals were fasted for 12 hours, and were sacrificed by excessive anesthesia with zoletil and ethyl ether. The experimental animals were opened, whole blood was collected from the portal vein, and plasma was separated. Plasma and tissue samples were stored at -80°C until use.

(2) Measurement of weight change according to diet control

The weight of the experimental animals was measured at a constant time every day for the entire experimental period, and the results are shown in FIG. 6. 6 is a diagram showing the change in body weight over time when the selected microorganisms are administered in obesity-induced mice. In FIG. 6, the horizontal axis represents time (weeks), and the vertical axis represents the percent weight gain ratio, that is (((experiment end time-high fat diet start date)/mouse weight at high fat diet start date) x100.

As shown in FIG. 6, the weight gain in the group fed the obesity-induced high fat diet was increased, and the weight gain rate was further increased in the group fed E. chlorase along with the high fat diet. In the case of the high-fat diet and the group receiving oral administration of E. chlorase and lactobacilli, there was no significant difference in dietary intake compared to the group inducing obesity with the high-fat diet and high-fat diet and E. chloracee, but the weight was significantly greater. Decreased. Therefore, in the case of the L. plantarum LMT1-48 administration group, it was confirmed that the function of promoting the weight gain rate by the obese bacteria E. chlorase was suppressed.

(3) Measurement of the volume ratio of adipose tissue

After the end of the 10 week experiment period, the change in body fat in the total body volume was measured for mice in the experimental animal group through PET-CT. In general, between lumbar vertebrae 1 and 5 is regarded as the abdominal region (L1 to L5) and measured, the results are shown in FIG. 7. 7 is a diagram showing a decrease in body fat volume when a selected microorganism is administered in an obesity-induced mouse. 7A is a view showing the total volume of body fat measured from lumbar spine No. 1 (L1) to lumbar spine No. 5 (L5). 7B is a diagram illustrating an example of a body fat scan area. As a scan area, L1 represents the upper lumbar region of No. 1 (Lumbar 1 proximal end), and L5 denotes the lower end of lumbar vertebrae No. 5 (Lumbar vertebrate distal end). Experimental animals actually photographed were photographed from L1 to L5 regions, and abdominal fat was shown in both visceral fat and subcutaneous fat in brown. 7C shows an animal showing an average weight loss in each experimental group among the scan photos of the lumbar spine 2 (L2) area among the regions between L1 and L5, and compares the scan photos of the animals at the same location (L2). As shown in FIG. 7, in the group (P3) orally administered E. chloroacet together with a high-fat diet after induction of obesity, the percentage of body fat was the highest among the total volume, and the high-fat diet and E. In the case of the oral administration group (P4), the body fat ratio was low.

As shown in Fig. 7c, the visceral fat and subcutaneous fat of the group (P3) administered orally with E. chlorase together with the high-fat diet group and the high-fat diet (P2) showed an increase in both visceral fat and subcutaneous fat, whereas L. plantarum. It can be seen that the LMT1-48 administration group (P4) exhibits an effect of inhibiting body fat accumulation in the abdominal region.

(4) Serum analysis

After the end of the 10-week experiment period, blood samples collected from each mouse were collected in tubes, and serum was separated by centrifugation. Total cholesterol, triglycerol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), leptin, and adiponectin contents were measured in the separated serum, and the results are shown in FIG. 8. Figure 8 is a case of administering selected microorganisms in obesity-induced mice, triglycerides in serum (Figure 8a), total cholesterol (Figure 8b), high-density lipoprotein (HDL) (Figure 8c), low-density lipoprotein (LDL) (Figure 8d) , Leptin (FIG. 8E), and adiponectin (FIG. 8F) is a diagram showing the results of measuring the content.

As shown in FIG. 8, the levels of cholesterol, high-density lipoprotein, leptin, and adiponectin were increased in the group fed the obesity-inducing high-fat diet, and cholesterol, triglycerol, high-density lipoprotein, and the group fed E. chlorase along with the high-fat diet were increased. The levels of leptin and adiponectin increased more. On the other hand, it can be seen that triglycerol and leptin are significantly reduced in the group administered orally with lactic acid bacteria. It has been found to be excellent in inhibiting the production of body fat, as well as inhibiting the secretion of hormones secreted from blood lipids and fats. In particular, leptin was found to significantly ( p <0.05) lower blood levels than in the high-fat diet group, suggesting that vital signs related to energy metabolism secreted from accumulated adipocytes were reduced. In the case of low-density lipoprotein, no effect was observed by E. chlorase and no effect by lactic acid bacteria was observed.

(5) Obesity bacteria Enterobacter Cloacee Caused by obesity C57BL Intestinal microbial change analysis in /6J mouse model

Before the end of the experiment in Example 2-3, the last feces were collected, and as for fecal samples, genomic DNA (gDNA) of microorganisms in feces was extracted using the UltraClean Fecal DNA Isolation Kit. The 16s rRNA gene was amplified by PCR using the extracted gDNA. The primers used at this time were barcoded primers 519F: SEQ ID NO: 24 and 806R: SEQ ID NO: 25, and the DNA product has a nucleotide sequence of about 450bp. Pyrosequencing was performed with the DNA product to analyze the intestinal flora distribution. Pyrosequencing was performed using Macrogen's MiSeq™ platform (Illumina, San Diego, USA).

The results of the analysis in which the distribution of intestinal microorganisms is classified into the river level are shown in Table 10 below.

P1 P2 P3 P4 Bacteria 0.08% 0.06% 0.07% 0.13% Actinobacteria 0.95% 0.72% 0.40% 0.35% Bacteroidetes 0.43% 1.87% 2.13% 0.00% Bacteroidia 73.30% 64.36% 61.80% 53.22% Flavobacteriia 0.01% 0.05% 0.03% 0.03% Deferribacteres 0.05% 0.00% 0.00% 0.19% Alphaproteobacteria 0.75% 0.04% 0.06% 0.01% Deltaproteobacteria 0.00% 0.00% 0.00% 0.00% Gammaproteobacteria 0.12% 0.11% 0.23% 2.73% Mollicutes 0.11% 2.91% 1.87% 3.28% Verrucomicrobiae 0.00% 0.00% 0.00% 1.62% Candidatus Saccharibacteria 1.87% 1.67% 1.27% 0.51% Chloroplast 0.03% 0.02% 0.00% 0.00% Firmicutes 0.33% 0.43% 0.36% 0.70% Bacilli 7.80% 2.64% 1.87% 2.53% Clostridia 13.69% 24.89% 29.76% 34.53% Erysipelotrichia 0.49% 0.23% 0.14% 0.17%

9 is a diagram showing the result of classifying the intestinal microorganisms in the feces at the level of the river when the selected microorganisms are administered in obesity-induced mice. As shown in Fig. 9, it was found that Bacteroidia , Erysipelotrichi , and Clostridia are dominant bacteria . As a result of classifying the intestinal microbes changed due to inhibition of the growth of obesity bacteria after ingestion of lactic acid bacteria, it was confirmed that the diversity of the intestinal microbes increased in the experimental group P5 compared to the control groups P2 and P3 that consumed the high fat diet. In addition, as a result of analysis classified by species level to confirm the presence or absence of intestinal lactic acid bacteria L. plantarum LMT 1-48 administered to the experimental group, it was confirmed that the ingested lactic acid bacteria species remained. Table 11 is a table showing the residual levels in feces of the administered microorganism.

Confirmation of residual lactic acid bacteria (Species level) Bell P1 P2 P3 P4 L. plantarum (LMT 1-48) 0 0 0 0.22

Example 3. Selected L. plantarum LMT 1-48 confirmation Marker And

In this example, a gene specifically present in the selected microorganism was identified, and it was confirmed whether the microorganism could be identified from other lactic acid bacteria.

First, high-quality gDNA was extracted from the microorganism using a Genomic DNA prep kit, solution type (Biofact, GD262-060). Next, for the extracted gDNA, sequence analysis of the complete genome was completed using the genome sequencing instrument Sequel II System (Pacific Bioscience), and Hiseq 2500 System (Illumina) (hereinafter,'experimental group genome' It is called).

Next, the NCBI genome, specifically Accession Number: PRJNA505395, was used as a reference genome by collecting genomes of 235 L. plantarum strains of the same species. The experimental group genomes and 235 standard genomes were registered in the analysis tool BIOiPLUG (Chunlab) for whole genome analysis and comparative genome analysis, respectively. A strain-specific coding sequence (CDS) was investigated by comparing the experimental group genome and the standard genome. Among the selected strain-specific CDS, the following three conditions were selected for verification using the PCR technique. ① Genes with short length are excluded. ② Genes having sequences similar to other strains were excluded through NCBI nucleotide-blast. ③ Genes that do not have a function were excluded. As a result, 10 genes were selected as unique-genes present in LMT 1-48.

For the selected strain-specific 10 coding sequences or genes, 10 primers per gene were prepared using NCBI primer-blast. PCR experiments were performed using the prepared primers. At this time, the template is the experimental group genome, its same species strain (Lactobacillus plantarum LMT1-4, LMT1-5, LMT1-7, LMT1-14, LMT1-16, LMT1-17, LMT1-33, LMT1-50, LMT1-52, LMT1-77, LMT2-10, LMT3-33, LMT3-61, LMT6-4, LMT13-14, LMT13-80, LMT13-116, LMT14-19, LMT17-5, LMT17-31, LMT17-32, LMT18- 20, LMT18-27, LMT18-30), type strain (Lactobacillus plantarum KCTC3108 ^T ) and other products, specifically Daewon Pharmaceutical, Jang Daewon L. plantarum CLP0611; Dsimone, Dsimone, L. plantarum DSM24730; I-Heal, I-Heal Venus Lactobacillus, L. plantarum MG989; The genomes of Pulmuone, plant lactic acid bacteria, and PMO08 were used. Specifically, a template DNA was prepared by resuspending microorganisms on a single colony in 200 μL 1X PBS buffer. Next, the template, distilled water, and primer were mixed with 2x TOPsimple DyeMIX-nTaq (Enzynomics). PCR was performed at the following temperature cycles in consideration of the annealing temperature provided to the prepared primers: pre-denaturation; 95°C, 10 min, denaturation; 95° C., 1 min, annealing; Conditional, 30sec, extension; 72℃, 1min, 30 cycle.

FIG. 10 shows the three species-gene-specific primer set positions selected in this example, PCR was performed using the same, and the result of electrophoresis analysis of the product. As shown in FIG. 10, LPL148_03160_1, LPL148_03166_6, and LPL148_03166_8 were specifically amplified products found in LMT 1-48 strains.

As a result, two specific genes specific to LMT 1-48 and three primer sets specific to this gene were selected. One of the two specific genes has the nucleotide sequence of SEQ ID NO: 1 and appears to encode a polypeptide having the amino acid sequence of SEQ ID NO: 9. This gene was named LPL148_03160, and its product is presumed to function as a histidine utilization repressor. As the LPL148_03160 gene-specific primer set, a primer set (LPL148_03160_1) of SEQ ID NOs: 2 and 3 was prepared.

The other has the nucleotide sequence of SEQ ID NO: 4 and appears to encode a polypeptide having the amino acid sequence of SEQ ID NO: 10. This gene was named LPL148_03166, and its product is presumed to function as mannose-6-phosphate isomerase. As LPL148_03166 gene-specific primer sets, primer sets of SEQ ID NOs: 5 and 6 (LPL148_03166_6) and primer sets of SEQ ID NOs: 7 and 8 (LPL148_03166_8) were prepared.

Korea Research Institute of Bioscience and Biotechnology KCTC13024BP 20160513

SEQUENCE LISTING <110> Medytox Inc., Ltd. <120> Microorganism capable of reducing lipid content in a subject, composition or kit for identifying thefore, and use thereof <130> PN130581KR <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 738 <212> DNA <213> Lactobacillus plantarum LMT 1-48 <400> 1 atgagtgagc cgaaatacaa ggcaattgaa aatgatttac gggccgccat cgagaacggt 60 acgtacaaag agggcgacct gattcccaaa gagatggagt tggtcgccca gtacgcggtc 120 agccgcccca cggtacgcca ggccatcgcg aatttagtta atgatggact attgcagaag 180 aagcggcatg ttgggaccgt ggtgcgggcc aataagattg gccaggagtt cacacatgtc 240 atcgaaagtt atgataagga aatgcgggcc aaagggttaa caacttctac acaggtattg 300 gcgtaccagg atgaacccgc cacggctgag gtggccgaga aactcggcct gcatgagggt 360 gatcaggtat ataaactgat ccgcttacgc tttgcggggg agcagcccat tgtgctagtt 420 acgagctacc taccctacgc tgtattgccc ggctttggta gtatcgattt caaccacacg 480 tcgttataca acgagctcaa taaggccgga aagccggtca cgcacgttca ccgtaagctt 540 gaggtagttt caggggatga gactacgagc gcgctgttga atgtgccaac gggagcacca 600 ctgttctact tccgtacgca gggatcgtgt ccagacggcg agattgttga gtattcgcaa 660 gctaagtatc ggggcgactc caactacttt atttttgatt tggataagag tgcgaatgat 720 aatgagttga gattgtag 738 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> LPL148_03160 primer Forward <400> 2 acggtacgta caaagagggc 20 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> LPL148_03160 primer Reverse <400> 3 tggagtcgcc ccgatactta 20 <210> 4 <211> 930 <212> DNA <213> Lactobacillus plantarum LMT 1-48 <400> 4 atgctgatcc taacaccatt ttccgcccca cggatttggg gcacagagcg tttacgtgag 60 tatggggcag acaaatcaac aacgggctcg gtatattcgg tcgcaggcac cgaccaactc 120 gacgtcgtgg ccctcgacac agtaaacggt gaccaatcct ctttgaagac aatagttcaa 180 agtaaccccg agcgattcgg gctacttcct ggcgaagaat accctatcat cgtagacatg 240 ctgggtgcag acgctgacct gagcattcaa gtgcacccca ccgatcagtt tgcacgttcc 300 caaggtttga attatgggaa aagtgagtca tgggtattcc tcactgcccc cacatccggt 360 acggtgtatt ctggtctgcg taatccccaa tacaaagttc aacctgacga atttcggtca 420 cacccactta cgatagtaga tgagcaaccg gttgagattg gcgactacgc ctttgttcct 480 agcggcaccg tgcacgcaat tcgggctggc agtctggtct acgaaattca acaatcgacc 540 gacattacgt accggctata cgattacaac cggccgggac taaatggaca gccccgtgcc 600 ttggacgttg aagacgccct acaaaacgta gtcaccacaa gcaaggtaca gattcaacac 660 tggggagcaa ctgcaagcgt tacggaagcg gcctaccatc tgagtaagct tacaataagc 720 ggcaagtatg actacgttgc cccggccggc gtggctacta gcgttaccgt ggtagctggc 780 gccatcacgg ttggtgacca taccattcac caagggggca gtatcatcgt cccgcccggc 840 gaacgtacgg caattcaggg tagcgccacc atcattgctg cgaccccgcg ggcgtactgg 900 cgtgaacaca cagaaactat ctcaatttag 930 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> >LPL148_03166 (1) forward <400> 5 gtggccctcg acacagtaaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial <220> <223>> LPL148_03166 (1) reverse <400> 6 cgatgatact gcccccttgg 20 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <223> LPL148_03166 (2) forward <400> 7 tttggggcac agagcgttta 20 <210> 8 <211> 20 <212> DNA <213> Artificial <220> <223> LPL148_03166 (2) reverse <400> 8 gcccccttgg tgaatggtat 20 <210> 9 <211> 245 <212> PRT <213> Lactobacillus plantarum LMT 1-48 <400> 9 Met Ser Glu Pro Lys Tyr Lys Ala Ile Glu Asn Asp Leu Arg Ala Ala 1 5 10 15 Ile Glu Asn Gly Thr Tyr Lys Glu Gly Asp Leu Ile Pro Lys Glu Met 20 25 30 Glu Leu Val Ala Gln Tyr Ala Val Ser Arg Pro Thr Val Arg Gln Ala 35 40 45 Ile Ala Asn Leu Val Asn Asp Gly Leu Leu Gln Lys Lys Arg His Val 50 55 60 Gly Thr Val Val Arg Ala Asn Lys Ile Gly Gln Glu Phe Thr His Val 65 70 75 80 Ile Glu Ser Tyr Asp Lys Glu Met Arg Ala Lys Gly Leu Thr Thr Ser 85 90 95 Thr Gln Val Leu Ala Tyr Gln Asp Glu Pro Ala Thr Ala Glu Val Ala 100 105 110 Glu Lys Leu Gly Leu His Glu Gly Asp Gln Val Tyr Lys Leu Ile Arg 115 120 125 Leu Arg Phe Ala Gly Glu Gln Pro Ile Val Leu Val Thr Ser Tyr Leu 130 135 140 Pro Tyr Ala Val Leu Pro Gly Phe Gly Ser Ile Asp Phe Asn His Thr 145 150 155 160 Ser Leu Tyr Asn Glu Leu Asn Lys Ala Gly Lys Pro Val Thr His Val 165 170 175 His Arg Lys Leu Glu Val Val Ser Gly Asp Glu Thr Thr Ser Ala Leu 180 185 190 Leu Asn Val Pro Thr Gly Ala Pro Leu Phe Tyr Phe Arg Thr Gln Gly 195 200 205 Ser Cys Pro Asp Gly Glu Ile Val Glu Tyr Ser Gln Ala Lys Tyr Arg 210 215 220 Gly Asp Ser Asn Tyr Phe Ile Phe Asp Leu Asp Lys Ser Ala Asn Asp 225 230 235 240 Asn Glu Leu Arg Leu 245 <210> 10 <211> 309 <212> PRT <213> Lactobacillus plantarum LMT 1-48 <400> 10 Met Leu Ile Leu Thr Pro Phe Ser Ala Pro Arg Ile Trp Gly Thr Glu 1 5 10 15 Arg Leu Arg Glu Tyr Gly Ala Asp Lys Ser Thr Thr Gly Ser Val Tyr 20 25 30 Ser Val Ala Gly Thr Asp Gln Leu Asp Val Val Ala Leu Asp Thr Val 35 40 45 Asn Gly Asp Gln Ser Ser Leu Lys Thr Ile Val Gln Ser Asn Pro Glu 50 55 60 Arg Phe Gly Leu Leu Pro Gly Glu Glu Tyr Pro Ile Ile Val Asp Met 65 70 75 80 Leu Gly Ala Asp Ala Asp Leu Ser Ile Gln Val His Pro Thr Asp Gln 85 90 95 Phe Ala Arg Ser Gln Gly Leu Asn Tyr Gly Lys Ser Glu Ser Trp Val 100 105 110 Phe Leu Thr Ala Pro Thr Ser Gly Thr Val Tyr Ser Gly Leu Arg Asn 115 120 125 Pro Gln Tyr Lys Val Gln Pro Asp Glu Phe Arg Ser His Pro Leu Thr 130 135 140 Ile Val Asp Glu Gln Pro Val Glu Ile Gly Asp Tyr Ala Phe Val Pro 145 150 155 160 Ser Gly Thr Val His Ala Ile Arg Ala Gly Ser Leu Val Tyr Glu Ile 165 170 175 Gln Gln Ser Thr Asp Ile Thr Tyr Arg Leu Tyr Asp Tyr Asn Arg Pro 180 185 190 Gly Leu Asn Gly Gln Pro Arg Ala Leu Asp Val Glu Asp Ala Leu Gln 195 200 205 Asn Val Val Thr Thr Ser Lys Val Gln Ile Gln His Trp Gly Ala Thr 210 215 220 Ala Ser Val Thr Glu Ala Ala Tyr His Leu Ser Lys Leu Thr Ile Ser 225 230 235 240 Gly Lys Tyr Asp Tyr Val Ala Pro Ala Gly Val Ala Thr Ser Val Thr 245 250 255 Val Val Ala Gly Ala Ile Thr Val Gly Asp His Thr Ile His Gln Gly 260 265 270 Gly Ser Ile Ile Val Pro Pro Gly Glu Arg Thr Ala Ile Gln Gly Ser 275 280 285 Ala Thr Ile Ile Ala Ala Thr Pro Arg Ala Tyr Trp Arg Glu His Thr 290 295 300 Glu Thr Ile Ser Ile 305 <210> 11 <211> 1159 <212> DNA <213> Lactobacillus plantarum LMT1-48 <400> 11 taataccgca taacaacttg gaccgcatgg tccgagcttg aaagatggct tcggctatca 60 cttttggatg gtcccgcggc gtattagcta gatggtgggg taacggctca ccatggcaat 120 gatacgtagc cgacctgaga gggtaatcgg ccacattggg actgagacac ggcccaaact 180 cctacgggag gcagcagtag ggaatcttcc acaatggacg aaagtctgat ggagcaacgc 240 cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt gttaaagaag aacatatctg 300 agagtaactg ttcaggtatt gacggtattt aaccagaaag ccacggctaa ctacgtgcca 360 gcagccgcgg taatacgtag gtggcaagcg ttgtccggat ttattgggcg taaagcgagc 420 gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc aaccgaagaa gtgcatcgga 480 aactgggaaa cttgagtgca gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg 540 tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac tgacgctgag 600 gctcgaaagt atgggtagca aacaggatta gataccctgg tagtccatac cgtaaacgat 660 gaatgctaag tgttggaggg tttccgccct tcagtgctgc agctaacgca ttaagcattc 720 cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag 780 cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac cttaccaggt cttgacatac 840 tatgcaaatc taagagatta gacgttccct tcggggacat ggatacaggt ggtgcatggt 900 tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt 960 atcagttgcc agcattaagt tgggcactct ggtgagactg ccggtgacaa accggaggaa 1020 ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1080 ggatggtaca acgagttgcg aactcgcgag agtaagctaa tctcttaaag ccattctcag 1140 ttcggattgt aggctgcaa 1159 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agagtttgat cmtggctcag 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggttaccttg ttacgactt 19 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gcatggtgcc ttcgctga 18 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tggcatctct gtgtcaacca tg 22 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 caagaacagc aacgagtacc g 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gtcactggtc aactccagca c 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgggttctag ccagcagagt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 accaccagag accgttatgc 20 <210> 20 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 agtgggcttt gccacaa 17 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ggtgatttca tcgaattcca 20 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 aacgacccct tcattgac 18 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tccacgacat actcagcac 19 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g, or t <400> 24 cctacgggng gcwgcag 17 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 gactachvgg gtatctaatc c 21

Claims (10)

LPL148-03160 gene of SEQ ID NO: 1, and LPL148-03166 gene of SEQ ID NO: 4, comprising the step of detecting one or more genes selected from the group consisting of, Lactobacillus plantarum capable of reducing fat content in adipocytes ( Lactobacillus plantarum) LMT1-48 (accession number: KCTC13024BP) method for identifying. delete The method of claim 1, wherein the detecting comprises a polynucleotide of SEQ ID NO: 2 and a polynucleotide of SEQ ID NO: 3; The polynucleotide of SEQ ID NO: 5 and the polynucleotide of SEQ ID NO: 6; And performing a polymerase chain reaction (PCR) using one or more primer sets selected from the group consisting of a polynucleotide of SEQ ID NO: 7 and a polynucleotide of SEQ ID NO: 8. delete LPL148-03160 gene of SEQ ID NO: 1, and LPL148-03166 gene of SEQ ID NO: 4, comprising a reagent for detecting at least one gene selected from the group consisting of, Lactobacillus plantarum capable of reducing fat content in adipocytes ( Kit for identifying Lactobacillus plantarum) LMT1-48 (accession number: KCTC13024BP). delete The method according to claim 5, A kit for use in detecting a gene by performing a polymerase chain reaction (PCR), the detecting reagent comprises a polynucleotide of SEQ ID NO: 2 and a polynucleotide of SEQ ID NO: 3; The polynucleotide of SEQ ID NO: 5 and the polynucleotide of SEQ ID NO: 6; And a polynucleotide of SEQ ID NO: 7 and a polynucleotide of SEQ ID NO: 8; a kit comprising one or more primer sets selected from the group consisting of. delete delete delete

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